Interferon is a glycoprotein produced by living cells in defense against virus infection. Its chemical structure may vary slightly depending upon the cell type that is used for its production. This interferon has various biochemical activities, such as antiviral, antiprotozoal, cell growth inhibitory and immunosuppressive activities and may, therefore, be applied successfully in medicine. For a general review of the present knowledge of interferon, reference may be made to the book "Interferons and their actions" by W. E. Stewart II, CRC Press, Inc., Cleveland, Ohio.
For medical application in human patients, interferon should be prepared from human cells and the following methods for this purpose are now in use:
1. Production by infection of freshly collected leukocytes from human blood donors with Sendai-virus. This results in the so-called leukocyte interferon as described by Cantell et al, In vitro, 3, 35-38, (1974).
2. Production on cultivated diploid human fibroblast cells with the aid of a so-called "Poly-I:C-superinduction schedule". The resulting fibroblast interferon differs from leukocyte interferon by several biological and physiochemical criteria (see A. Billiau et al, J. Gen. Virol., 19, 1-8, (1973)).
3. Production in lymphoblastoid cell lines using a viral interferon inducer. The resulting lymphoblast interferon has a strong resemblance to leukocyte interferon (see Strander et al, J. Clin. Microbiol., 1, 116-117, (1975)).
The present invention relates to human fibroblast interferon, that is interferon produced by the second of the above-mentioned methods, and more specifically, it relates to a method for the purification of such interferon.
Purification of interferon is necessary both for studies on the chemical character of interferon and for clinical application thereof, since a crude interferon solution may contain contaminating proteins that have a negative effect on the results of such studies and application.
For both purposes, rather large amounts of purified interferon are needed. Although many techniques for partial purification and a few techniques for complete purification of interferon have been described, all of them suffer from deficiencies. These deficiencies are in general, either the necessity of using complex adsorbents or reagents, or the use of a multiple step procedure which is not applicable to large scale production, or the fact that only a small fraction of the total initial interferon activity is recovered in purified form.
Therefore, a need exists for a purification method of interferon, and especially a purification method of human fibroblast interferon, which leads to a high recovery of activity in purified form, which does not require complex reagents or a complexity of steps, and which is capable of being used on a rather large scale.
As a result of extensive research, it has now been found that a high recovery of interferon activity in the form of completely purified interferon may be obtained by treating human fibroblast interferon with a simple two-step purification method which is a combination of two earlier known methods. This two-step purification method comprises (a) subjecting an aqueous interferon solution to chromatography on porous glass beads, and (b) subjecting the resulting aqueous interferon solution to chromatography on immobilized zinc chelate.
If, in the first step, an interferon solution containing contaminating proteins is contacted with porous glass beads at neutral or slightly alkaline pH, the interferon will be selectively adsorbed onto these glass beads and the bulk of contaminating proteins will remain in solution and may be washed away. The adsorbed interferon may thereafter be eluted from the glass beads at an acidic pH.
If thereupon, in the second step, the eluted interferon solution is contacted with an immobilized zinc chelate gel at neutral or slightly alkaline pH, the interferon will be selectively adsorbed onto this zinc chelate and contaminating proteins that might still be present will remain in solution and may be washed away. The adsorbed interferon may thereafter be eluted from the zinc chelate at acidic pH and will result in an end product of extremely high purity.
By using this two-step purification method, a high degree of purification may be reached since the end product is of such high purity that it may be considered to be substantially completely pure. Further, a high recovery of initial interferon activity may be achieved. This recovery will be about 50-70% in the first step, and 90-95% in the second step, thus resulting in an overall recovery of about 45-67%.
A special advantage is that the end product of the invented purification method will be free of any skin reactive agent. The products of earlier purification methods always incited skin reactions upon clinical application to patients but it has appeared that the product of the present invention will not incite such reactions and this is quite important for clinical use.
A further advantage is that the reagents are easily available and may be used many times in succession since both the glass beads and the immobilized zinc chelate may be reclaimed or regenerated after use. Further, the invented method uses only two steps and all these facts result in a simple method that can be used on a rather large scale.
Thus, the invention provides a method for the purification of interferon, which comprises (a) subjecting an aqueous solution of human fibroblast interferon to chromatography on porous glass beads, and (b) subjecting the resulting interferon solution to chromatography on immobilized zinc chelate.
It should be noted here that both steps of the invented method are known individually and have been used earlier for purification of human fibroblast interferon. Compare A. Billiau et al, Antimicrobial Agents and Chemotherapy, 16, 49-55 (1979) for the first step, and V. G. Edy et al, J. Biol. Chem. 252, 5934-5935 (1977) for the second step. At the time of these publications, however, the said steps were used quite independently and there was nothing to suggest that a substantially complete purification would be brought about by combining these steps in a simple two-step method.
Moreover, several other methods are known for purification of interferon and it would be impossible to predict that a combination of the aforesaid two steps without necessity of adding further steps, would lead to the desired result. Further, the absence of any skin reactivity in the end products of the invented method can be termed surprising and unexpected because until now, all purified fibroblast interferon showed such skin reactivity upon clinical application to patients (compare A. Billiau et al, Antimicrobial Agents and Chemotherapy, 16, 56-63 (1979)).
Furthermore, it should be noted that the combination of both steps in a two-step method has led to a slight modification of the first step; as far as the eluate of the glass beads is concerned. In the above-mentioned first Billiau paper, this eluate was dialyzed against polyethyleneglycol in a sodium acetate buffer in order to prepare it for lyophilization and clinical use. In the present invention, however, such eluate will be dialyzed against a phosphate buffer in order to prepare it for chromatography in the next purification step, as will be described later on in this specification.